In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles.

RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.

Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.

Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p-/-) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p-/- mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.

Tuning the size, shape and structure of RNA nanoparticles for favorable cancer targeting and immunostimulation.

The past decade has shown exponential growth in the field of RNA nanotechnology. The rapid advances of using RNA nanoparticles for biomedical applications, especially targeted cancer therapy, suggest its potential as a new generation of drug. After the first milestone of small molecule drugs and the second milestone of antibody drugs, it was predicted that RNA drugs, either RNA itself or chemicals/ligands that target RNA, will be the third milestone in drug development. Thus, a comprehensive assessment of the current therapeutic RNA nanoparticles is urgently needed to meet the drug evaluation criteria. Specifically, the pharmacological and immunological profiles of RNA nanoparticles need to be systematically studied to provide insights in rational design of RNA-based therapeutics. By virtue of its programmability and biocompatibility, RNA molecules can be designed to construct sophisticated nanoparticles with versatile functions/applications and highly tunable physicochemical properties. This intrinsic characteristic allows the systemic study of the effects of various properties of RNA nanoparticles on their in vivo behaviors such as cancer targeting and immune responses. This review will focus on the recent progress of RNA nanoparticles in cancer targeting, and summarize the effects of common physicochemical properties such as size and shape on the RNA nanoparticles' biodistribution and immunostimulation profiles. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Diagnostic Tools > in vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression.

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery.

A novel modified nucleic acid nanoparticle harboring an annexin A2 aptamer for ovarian cancer cell targeting and a GC rich sequence for doxorubicin loading is designed and constructed. The system utilizes a highly stable three-way junction (3WJ) motif from phi29 packaging RNA as a core structure. A phosphorothioate-modified DNA aptamer targeting annexin A2, Endo28, was conjugated to one arm of the 3WJ. The pRNA-3WJ motif retains correct folding of attached aptamer, keeping its functions intact. It is of significant utility for aptamer-mediated targeted delivery. The DNA/RNA hybrid nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little accumulation in healthy organs 6 h post-injection. The Endo28-3WJ-Sph1/Dox intercalates selectively enhanced toxicity to annexin A2 positive ovarian cancer cells in vitro. The constructed RNA/DNA hybrid nanoparticles can potentially enhance the therapeutic efficiency of doxorubicin at low doses for ovarian cancer treatment through annexin A2 targeted drug delivery.

Development of Potent Antiviral Drugs Inspired by Viral Hexameric DNA-Packaging Motors with Revolving Mechanism.

The intracellular parasitic nature of viruses and the emergence of antiviral drug resistance necessitate the development of new potent antiviral drugs. Recently, a method for developing potent inhibitory drugs by targeting biological machines with high stoichiometry and a sequential-action mechanism was described. Inspired by this finding, we reviewed the development of antiviral drugs targeting viral DNA-packaging motors. Inhibiting multisubunit targets with sequential actions resembles breaking one bulb in a series of Christmas lights, which turns off the entire string. Indeed, studies on viral DNA packaging might lead to the development of new antiviral drugs. Recent elucidation of the mechanism of the viral double-stranded DNA (dsDNA)-packaging motor with sequential one-way revolving motion will promote the development of potent antiviral drugs with high specificity and efficiency. Traditionally, biomotors have been classified into two categories: linear and rotation motors. Recently discovered was a third type of biomotor, including the viral DNA-packaging motor, beside the bacterial DNA translocases, that uses a revolving mechanism without rotation. By analogy, rotation resembles the Earth's rotation on its own axis, while revolving resembles the Earth's revolving around the Sun (see animations at http://rnanano.osu.edu/movie.html). Herein, we review the structures of viral dsDNA-packaging motors, the stoichiometries of motor components, and the motion mechanisms of the motors. All viral dsDNA-packaging motors, including those of dsDNA/dsRNA bacteriophages, adenoviruses, poxviruses, herpesviruses, mimiviruses, megaviruses, pandoraviruses, and pithoviruses, contain a high-stoichiometry machine composed of multiple components that work cooperatively and sequentially. Thus, it is an ideal target for potent drug development based on the power function of the stoichiometries of target complexes that work sequentially.

Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting.

RNA tetrahedral nanoparticles with two different sizes are successfully assembled by a one-pot bottom-up approach with high efficiency and thermal stability. The reported design principles can be extended to construct higher-order polyhedral RNA architectures for various applications such as targeted cancer imaging and therapy.

Controllable self-assembly of RNA dendrimers.

We report programmable self-assembly of branched, 3D globular, monodisperse and nanoscale sized dendrimers using RNA as building blocks. The central core and repeating units of the RNA dendrimer are derivatives of the ultrastable three-way junction (3WJ) motif from the bacteriophage phi29 motor pRNA. RNA dendrimers were constructed by step-wise self-assembly of modular 3WJ building blocks initiating with a single 3WJ core (Generation-0) with overhanging sticky end and proceeding in a radial manner in layers up to Generation-4. The final constructs were generated under control without any structural defects in high yield and purity, as demonstrated by gel electrophoresis and AFM imaging. Upon incorporation of folate on the peripheral branches of the RNA dendrimers, the resulting constructs showed high binding and internalization into cancer cells. RNA dendrimers are envisioned to have a major impact in targeting, disease therapy, molecular diagnostics and bioelectronics in the near future. FROM THE CLINICAL EDITOR: Dendrimers are gaining importance as a carrier platform for diagnosis and therapeutics. The authors here reported building of their dendrimer molecules using RNA as building blocks. The addition of folate also allowed recognition and subsequent binding to tumor cells. This new construct may prove to be useful in many clinical settings.

Discovery of a new method for potent drug development using power function of stoichiometry of homomeric biocomplexes or biological nanomotors.

INTRODUCTION: Multidrug resistance and the appearance of incurable diseases inspire the quest for potent therapeutics. AREAS COVERED: We review a new methodology in designing potent drugs by targeting multi-subunit homomeric biological motors, machines or complexes with Z > 1 and K = 1, where Z is the stoichiometry of the target, and K is the number of drugged subunits required to block the function of the complex. The condition is similar to a series electrical circuit of Christmas decorations: failure of one light bulb causes the entire lighting system to lose power. In most multi-subunit, homomeric biological systems, a sequential coordination or cooperative action mechanism is utilized, thus K equals 1. Drug inhibition depends on the ratio of drugged to non-drugged complexes. When K = 1, and Z > 1, the inhibition effect follows a power law with respect to Z, leading to enhanced drug potency. The hypothesis that the potency of drug inhibition depends on the stoichiometry of the targeted biological complexes was recently quantified by Yang-Hui's Triangle (or binomial distribution), and proved using a highly sensitive in vitro phi29 viral DNA packaging system. Examples of targeting homomeric bio-complexes with high stoichiometry for potent drug discovery are discussed. EXPERT OPINION: Biomotors with multiple subunits are widespread in viruses, bacteria and cells, making this approach generally applicable in the development of inhibition drugs with high efficiency.

New approach to develop ultra-high inhibitory drug using the power function of the stoichiometry of the targeted nanomachine or biocomplex.

AIMS: To find methods for potent drug development by targeting to biocomplex with high copy number. METHODS: Phi29 DNA packaging motor components with different stoichiometries were used as model to assay virion assembly with Yang Hui's Triangle [Formula: see text], where Z = stoichiometry, M = drugged subunits per biocomplex, p and q are the fraction of drugged and undrugged subunits in the population. RESULTS: Inhibition efficiency follows a power function. When number of drugged subunits to block the function of the complex K = 1, the uninhibited biocomplex equals q(z), demonstrating the multiplicative effect of stoichiometry on inhibition with stoichiometry 1000 > 6 > 1. Complete inhibition of virus replication was found when Z = 6. CONCLUSION: Drug inhibition potency depends on the stoichiometry of the targeted components of the biocomplex or nanomachine. The inhibition effect follows a power function of the stoichiometry of the target biocomplex.

Construction of RNA-Quantum Dot Chimera for Nanoscale Resistive Biomemory Application.

RNA nanotechnology offers advantages to construct thermally and chemically stable nanoparticles with well-defined shape and structure. Here we report the development of an RNA-QD (quantum dot) chimera for resistive biomolecular memory application. Each QD holds two copies of the pRNA three-way junction (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor. The fixed quantity of two RNAs per QD was achieved by immobilizing the pRNA-3WJ with a Sephadex aptamer for resin binding. Two thiolated pRNA-3WJ serve as two feet of the chimera that stand on the gold plate. The RNA nanostructure served as both an insulator and a mediator to provide defined distance between the QD and gold. Immobilization of the chimera nanoparticle was confirmed with scanning tunneling microscopy. As revealed by scanning tunneling spectroscopy, the conjugated pRNA-3WJ-QD chimera exhibited an excellent electrical bistability signal for biomolecular memory function, demonstrating great potential for the development of resistive biomolecular memory and a nano-bio-inspired electronic device for information processing and computing.

RNA Nanoparticles Derived from Three-Way Junction of Phi29 Motor pRNA Are Resistant to I-125 and Cs-131 Radiation.

Radiation reagents that specifically target tumors are in high demand for the treatment of cancer. The emerging field of RNA nanotechnology might provide new opportunities for targeted radiation therapy. This study investigates whether chemically modified RNA nanoparticles derived from the packaging RNA (pRNA) three-way junction (3WJ) of phi29 DNA-packaging motor are resistant to potent I-125 and Cs-131 radiation, which is a prerequisite for utilizing these RNA nanoparticles as carriers for targeted radiation therapy. pRNA 3WJ nanoparticles were constructed and characterized, and the stability of these nanoparticles under I-125 and Cs-131 irradiation with clinically relevant doses was examined. RNA nanoparticles derived from the pRNA 3WJ targeted tumors specifically and they were stable under irradiation of I-125 and Cs-131 with clinically relevant doses ranging from 1 to 90 Gy over a significantly long time up to 20 days, while control plasmid DNA was damaged at 20 Gy or higher.

Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

RNA as a stable polymer to build controllable and defined nanostructures for material and biomedical applications.

The value of polymers is manifested in their vital use as building blocks in material and life sciences. Ribonucleic acid (RNA) is a polynucleic acid, but its polymeric nature in materials and technological applications is often overlooked due to an impression that RNA is seemingly unstable. Recent findings that certain modifications can make RNA resistant to RNase degradation while retaining its authentic folding property and biological function, and the discovery of ultra-thermostable RNA motifs have adequately addressed the concerns of RNA unstability. RNA can serve as a unique polymeric material to build varieties of nanostructures including nanoparticles, polygons, arrays, bundles, membrane, and microsponges that have potential applications in biomedical and material sciences. Since 2005, more than a thousand publications on RNA nanostructures have been published in diverse fields, indicating a remarkable increase of interest in the emerging field of RNA nanotechnology. In this review, we aim to: delineate the physical and chemical properties of polymers that can be applied to RNA; introduce the unique properties of RNA as a polymer; review the current methods for the construction of RNA nanostructures; describe its applications in material, biomedical and computer sciences; and, discuss the challenges and future prospects in this field.

Binomial distribution for quantification of protein subunits in biological nanoassemblies and functional nanomachines.

Living systems produce ordered structures and nanomachines that inspire the development of biomimetic nanodevices such as chips, MEMS, actuators, sensors, sorters, and apparatuses for single-pore DNA sequencing, disease diagnosis, drug or therapeutic RNA delivery. Determination of the copy numbers of subunits that build these machines is challenging due to small size. Here we report a simple mathematical method to determine the stoichiometry, using phi29 DNA-packaging nanomotor as a model to elucidate the application of a formula ∑M=0(Z)((Z)M)p(Z-M)q(M), where p and q are the percentage of wild-type and inactive mutant in the empirical assay; M is the copy numbers of mutant and Z is the stoichiometry in question. Variable ratios of mutants and wild-type were mixed to inhibit motor function. Empirical data were plotted over the theoretical curves to determine the stoichiometry and the value of K, which is the number of mutant needed in each machine to block the function, all based on the condition that wild-type and mutant are equal in binding affinity. Both Z and K from 1-12 were investigated. The data precisely confirmed that phi29 motor contains six copies (Z) of the motor ATPase gp16, and K=1. From the clinical editor: To determine copy numbers of subunits that form nanomachines in living organisms is a daunting task due to the complexities and the inherently small sizes associated with such systems. In this paper, a simple mathematical method is described how to determine the stoichiometry of copies in biomimetic nanodevices, using phi29 DNA-packaging nanomotor as a model.

Stable RNA nanoparticles as potential new generation drugs for cancer therapy.

Human genome sequencing revealed that only ~1.5% of the DNA sequence coded for proteins. More and more evidence has uncovered that a substantial part of the 98.5% so-called "junk" DNAs actually code for noncoding RNAs. Two milestones, chemical drugs and protein drugs, have already appeared in the history of drug development, and it is expected that the third milestone in drug development will be RNA drugs or drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is unique in that its scaffold, ligand, and therapeutic component can all be composed of RNA. The special physicochemical properties lend to the delivery of siRNA, miRNA, ribozymes, or riboswitches; imaging using fluogenenic RNA; and targeting using RNA aptamers. With recent advances in solving the chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors with favorable pharmacokinetic and pharmacodynamic parameters without unwanted accumulation in normal organs. This review summarizes the key studies that have led to the detailed understanding of RNA nanoparticle formation as well as chemical and thermodynamic stability issue. The methods for RNA nanoparticle construction, and the current challenges in the clinical application of RNA nanotechnology, such as endosome trapping and production costs, are also discussed.

[Preparation of ATP-2Na loaded liposome and its effect on tissues energy state in myocardial ischemic mice].

The aim of this study is to improve liposome encapsulation efficiency of water soluble drug ATP-2Na with hydrophobic ion pairing method, and evaluate its effect on tissues energy state in myocardial ischemia mice. Ion pair complex of ATP-2Na with HTAB was prepared first; then the liposomes were prepared by ethanol injection method. The size and zeta potential of ATP-2Na liposome were investigated. Its effect on tissues energy state in myocardium ischemia mice was evaluated by detecting ATP-2Na concentration in tissues and blood after injection in comparison to ATP-2Na solution. The diameters and zeta potential of ATP-2Na liposomes were (144.0 +/- 2.7) nm and (+16.2 +/- 1.6) mV, respectively. The encapsulation efficiency was (85.02 +/- 2.31) %. The in vitro drug release pattern from liposomes matches with Weibull equation. Compared with ATP-2Na solution, ATP-2Na liposome increased the ATP concentration of blood in myocardial ischemic mice very significantly; compared with blank, ATP-2Na liposome increased ATP content of myocardium and liver in myocaridal ischemic mice significantly, but ATP-2Na solution didn't show this effect. ATP-2Na liposome might have an advantage in improving tissue energy state on myocaridal ischemic animals.